1. Field of the Invention
The present invention relates generally oncology and cancer susceptibility. More particularly, it concerns methods for assessing cancer susceptibility, particularly lung cancer susceptibility, to carcinogens in tobacco smoke and products.
2. Description of the Related Art
Lung cancer is the leading cause of cancer mortality in the United States, and there is an urgent need to improve outcome by identifying and validating markers to predict risk and allow early diagnosis (1). A crucial early event in carcinogenesis is the induction of the genomic instability phenotype, which enables an initiated cell to evolve into a cancer cell by achieving a greater proliferative capacity (2). It is well known that cancer results from an accumulation of multiple genetic changes that can be mediated through chromosomal changes and that have the potential to be cytogenetically detectable (3). It has been hypothesized that the level of genetic damage in peripheral blood lymphocytes reflects amount of damage in the precursor cells that lead to the carcinogenic process in target tissues (4). Evidence that cytogenetic biomarkers are positively correlated with cancer risk has been strongly validated in recent results from both cohort and nested case-control studies showing that chromosome aberrations are a marker of cancer risk (5-9) reflecting both the genotoxic effects of carcinogens and individual cancer susceptibility.
A number of epidemiologic studies employing a variety of measures of DNA repair capacity have been performed to compare cancer patients and healthy control subjects and thereby assess the role of repair capacity in cancer risk (19). Using a variety of in vitro assays, we have previously shown (41-45) that sensitivity to mutagens varies widely between subjects with and without cancer and that this variation translates into interindividual variability in susceptibility to in vitro carcinogenic challenge. The mutagen sensitivity assay measures indirectly an individual's DNA repair capacity from cellular damage remaining after an in vitro mutagenic exposure and subsequent recovery. This assay, developed by Hsu et al (46), reflects general and nonspecific impairment of the DNA repair machinery and host genomic stability.
More than 80% of lung cancers are attributed to tobacco exposure. However, since only a fraction of long-term smokers (˜15%) will develop lung cancer in their lifetime (16), it is proposed that genetic factors play a role in individual susceptibility. An individual's DNA repair capacity may play a significant role in modifying risk for cancer. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a strong pulmonary carcinogen and is particularly important in the induction of adenocarcinoma, now the leading lung cancer subtype in the United States (17). Studies on the metabolism of NNK have shown that it induces cross-links in DNA; interacts with DNA, forming different types of adducts; and increases the frequency of chromosome aberrations (18, 19). Hecht (20) reported that DNA adducts derived from NNK are present at a higher level in lung tissues from lung cancer patients than controls, and metabolites of NNK are found in the urine of people who use tobacco products or are exposed to environmental tobacco smoke. The repair kinetics for NNK-induced genetic damage have not been clearly elucidated but may involve several DNA-repair pathways, including base excision and nucleotide excision repair pathways (21).
Tobacco smoke contains an array of potent carcinogens including polycyclic aromatic hydrocarbons, aromatic amines and N-nitrosamines. Among the polycyclic aromatic hydrocarbons, benzo [a] pyrene has been the most extensively studied, and our research to date has focused largely on this carcinogen. Benzo [a] pyrene is an effective pulmonary carcinogen, inducing predominantly squamous cell carcinoma upon intratracheal instillation into rats and hamsters. Tobacco-specific nitrosamines are found in high concentrations in mainstream smoke (28). The most potent carcinogenic member of this group, as shown in experimental animals, is NNK (29). NNK induces lung adenocarcinoma independent of route of administration and in both susceptible and resistant strains of mice (30). The estimated NNK dose of lifetime smokers (2 packs per day for 40 years) is 1.6 mg NNK per kilogram of body weight (31) close to the lowest dose shown to induce lung tumors in rats 1.8 mg (32). The total level of NNK in mainstream smoke is 3 to 15 times that of Benzo [a] pyrene (33). Gender differences and DNA repair allelic variants, have been reported to modulate the effect of NNK-induced genotoxic damage using the conventional chromosome aberration assay (24, 25) or fluorescence in situ hybridization assay using chromosome 1-specific probes in healthy smokers and nonsmoker controls (26). In addition, NNK was shown to be a potent mutagen using both the Ames Salmonella assay and the micronucleus test in Swiss mice (34).
The CBMN assay is a genotoxicity assay that provides simultaneous information on a variety of chromosomal damage endpoints that reflect chromosomal breakage, chromosome rearrangements, and gene amplification. In the current study, the frequencies of MN, NPBs, and NBUDs were significantly higher in the lung cancer patients than in controls. Cheng et al reported similar results after evaluating the MN frequency in 42 patients with lung cancer and 55 controls (35). The significantly higher spontaneous MN levels observed in the cases suggest a higher background level of genetic instability in the cancer patients. The effect of smoking on MN frequency in peripheral blood lymphocytes has not been consistent across studies, which generally have been small and not properly designed to detect the effect of smoking as the main outcome measure (36-38). However, a pooled re-analysis of 24 databases (5710 subjects, of which 1409 were current smokers) from the HUMN Project revealed that MN frequency was not influenced by the number of cigarettes smoked per day among subjects occupationally exposed to genotoxic agents, whereas a U-shaped curve was observed for nonexposed smokers, with a significant increase of MN frequency in individuals smoking 30 cigarettes or more per day (frequency ratio [FR]=1.59, 95% CI=1.35-1.88) (39).
This assay has been used to study susceptibility to other mutagenic agents. Scott et al (47) showed that individuals who developed breast cancer and their relatives were more sensitive than controls to the DNA-damaging effect of ionizing radiation, as demonstrated by MN frequency. This sensitivity was observed in 10 of 11 cases of BRACA1 mutation carriers and was indicative of a defect in double-strand break repair (48) thus suggesting that this assay is not only useful as a marker of DNA damage but also as a means of measuring the DNA repair phenotype. Umegaki and Fenech (11) recently validated the use of NPBs as a biomarker of DNA damage in a human B lymphoblastoid cell line (WIL2-NS). NPB frequency in binucleated cells increased up to 20 fold in the WIL2-NS cells relative to control cells in response to agents known to induce DNA strand breaks; the effects were found to be dose-dependant. Crott et al (49) recently reported that the frequency of NPBs and NBUDs correlated significantly and negatively with folic acid concentrations, suggesting that these chromosomal endpoints may be induced by folic acid deficiency. In addition, Kimura et al (50) showed a significant impact of methylene-tetrahydrofolate reductase (MTHFR) C677T polymorphism and folic acid concentration on MN, NPBs and NBUDS in human lymphocytes. Recently, scientists associated with the present inventors reported that polymorphisms in genes involved in folate metabolism were associated with lung cancer risk, an effect that may be modulated by dietary nutrient intake (51).
However, there is currently no known or reported method for actually predicting a subject's susceptibility to the development of smoking or tobacco-related cancers. There is no doubt that the development of such an assay would be of great medical importance, indeed, a medical breakthrough.